Gene analyses using DNA chips, beads, or the like require immobilization of synthetic oligonucleotides, PCR products, or the like as probes on supports. Probes immobilized on such supports complementarily bind to target nucleic acids labeled with fluorescence or the like. Hence, target nucleic acids are retained on the supports. Through measurement of fluorescence intensity derived from such a label, target nucleic acid levels can be detected. In the case of a DNA chip, several thousand to hundreds of thousands of types of probe have already been immobilized at predetermined positions on a plane support, so that the expression levels of many types of gene can be determined simultaneously. Therefore, such a DNA chip is extremely useful in elucidation of complicated networks among genes. The use of such DNA chip is expected to be a powerful technique for genetic diagnosis.
For immobilization of synthetic oligonucleotide probes on a support, there are methods that involve directly synthesizing oligonucleotides on a support (Nucleic Acids Res., vol. 20, 1675-1678 (1992) and Trends Biotechnol., vol. 12, 19-26 (1994)), and methods that involve purifying synthesized oligonucleotides and then immobilizing them on a support, for example. A method known as an example of the latter method involves introducing functional groups such as an amino group during oligonucleotide probe synthesis, spotting the resultants, and then covalently binding these functional groups to functional groups with which a support has been coated, thereby achieving irreversible immobilization of the probes. Furthermore, a method that involves electrostatically binding oligonucleotide probes to a support coated with positively-charged poly-L-lysine or the like has also been reported. Since electrostatic binding depends on the negative charge of oligonucleotides, the shorter the chain lengths of oligonucleotide probes, the lower the immobilization efficiency. Therefore, a method that involves immobilizing oligonucleotides on a support via covalent bonding is broadly employed.
When an amino group is introduced at an end of an oligonucleotide, a technique using an aminating reagent bound to a straight-chain alkyl group is generally employed (JP Patent Publication (Kokai) No. 59-27900 A (1984) and JP Patent Publication (Kokai) No. 60-166694 A (1985)). Several methods for synthesizing such type of aminating reagent have been reported. An aminating reagent that is currently mainly used has a C6 straight-chain alkyl chain (Methods Enzymol, 168, 753-761 (1989) and Nucleic Acids Res, 14, 6227-6245 (1986)). However, when this aminating reagent is used, the condensation rate of an oligonucleotide and an aminating reagent is insufficient. Hence, short oligonucleotides into which no amino groups have been introduced are mixed in as byproducts. Moreover, a condensation reaction of each nucleotide, which is performed in an automatic DNA synthesizer, results in the generation of some amounts of unreacted products. Hence, there is a need to separate and purify a target aminated oligonucleotide from such impurities. However, purification of a target aminated oligonucleotide with high purity is accompanied not only by the problem of requiring excessive time, but also by the problem of the impossibility of obtaining sufficient yields. Furthermore, in the case of purification of short duration, a target aminated oligonucleotide with sufficient purity has been impossible to obtain. This may be due to the properties of protecting groups for amino groups.
As protecting groups for amino groups in commercially available aminating reagents, a monomethoxytrityl group that can be removed under acidic conditions and a trifluoroacetyl group that can be removed under alkaline conditions are used. A monomethoxytrityl group can be removed for deprotection under acidic conditions that should be strict conditions (e.g., 1 or more hours in 80% acetic acid). Even under such conditions, protecting groups cannot be completely removed. Hence, the use of a monomethoxytrityl group is problematic in that it results in low yields of aminated oligonucleotides. Such conditions are unfavorable for DNA that is easily damaged under acidic conditions. Furthermore, much time is required for purification under such conditions. Therefore, high-throughput purification of aminated oligonucleotides is difficult. Meanwhile, a trifluoroacetyl group is rapidly removed for deprotection under alkaline conditions. Since deprotection of nucleotide portions of oligonucleotides is carried out under alkaline conditions, amino groups are also deprotected during such deprotection. Thus, purification using the hydrophobicity of protecting groups cannot be carried out, unlike the case of a monomethoxytrityl group. Undesirable unaminated oligonucleotides and aminated oligonucleotides have almost the same hydrophobicity. Thus, it has been difficult to separate and purify oligonucleotides containing amino groups protected with trifluoroacetyl groups from undesirable unaminated oligonucleotides. Therefore, it has been desired to develop an aminated oligonucleotide that can be rapidly synthesized and purified with high purity.